The purpose of this research is to study the synthesis and secretion of the catecholamines, epinephrine and norepinephrine, from the adrenal medulla. In particular, the goals of this research are to elucidate the roles of cyclic adenosine 3':5'-monophosphate (cAMP), Ca2 ion, protein kinase activity, and protein phosphorylation in these processes. I have recently developed methods for studying catecholamine synthesis and secretion in cell suspensions prepared from normal adrenal glands and from a transplantable rat pheochromocytoma. These cells secrete catecholamine, and also increase catecholamine synthesis, in response to incubation in media containing high concentrations of K ion. In the proposed experiments, suspensions of pheochromocytoma cells and of normal chromaffin cells will be incubated with high K ion, cholera toxin, and acetylcholine, and the activity of cAMP-dependent protein kinase in the cells will be measured. In other experiments, cells will be incubated with 32Pi, and will then be exposed to these agents; phosphorylated proteins will be separated by electrophoresis, and the effects of these stimuli on the phosphorylation of endogenous chromaffin cell proteins will be determined. The involvement of cAMP and of Ca2 ion in the regulation of endogenous protein phosphorylation, and the correlation between protein phosphorylation and the systhesis and secretion of catecholamines will be investigated. Finally, the regulation of tyrosine 3-monooxygenase activity in pheochromocytoma cells in tissue culture will be studied.